Acid Sphingomyelinase, E.C. 3.1.4.12, (ASM) is a lysosomal phosphodiesterase enzyme that hydrolyzes sphingomyelin, a phospholipid storage substance found in the brain, liver, lungs, spleen and lymph-nodes, to ceramide and phosphocholine. Deficiencies in ASM activity result in the inability of the body to break down sphingomyelin, causing a form of the lysosomal storage disease termed Niemann-Pick disease.
Niemann-Pick disease is an inherited autosomal recessive lipid storage disorder characterized by excessive accumulation of sphingomyelin in the lysosomes of cells such as macrophages and neurons, which impairs normal cellular function. Niemann-Pick Type A is a rapidly progressive neurodegenerative disease in infants and typically results in death within two to three years of age. Niemann-Pick Type B results in the enlargement of the liver and spleen, and respiratory distress with death generally ensuing by early adulthood. These two forms of Niemann-Pick disease which are both associated with ASM deficiencies are referred to collectively herein as Niemann-Pick disease. Other types of Niemann-Pick disease, e.g. Type C, do not involve mutations to the ASM gene and are not directly attributable to the function of ASM.
Enzyme replacement therapy is a well-known treatment for lysosomal storage diseases. Enzyme replacement therapy attempts to supplement the deficient enzyme activity with exogenously supplied enzyme. In the case of enzyme replacement therapy for Niemann-Pick disease, the goal would be to enable the affected individual to process sphingomyelin and avoid its buildup within the lysosomes. To be effective, such therapy initially would require a sufficiently large amount of the replacement enzyme to break down the accumulated sphingomyelin as well as continued administration of replacement enzyme to avoid further accumulation of sphingomyelin.
ASM is a glycoprotein with six potential N-glycosylation sites encoded by the amino acid sequence (Schuchman, E. H. et al, (1991) J. Biol. Chem., Vol. 266, 8531-8539). Site-directed mutagenesis studies have shown that at least five of the six sites are utilized (Ferlinz, K., et al., (1997) Eur. J. Biochem. Vol. 243, 511-517). This study also found that elimination of the four sites nearest the N-terminus does not disrupt lysosomal targeting, processing, or enzymatic activity. It was shown, however, that removal of the two C-terminal N-glycosylation sites results in either rapid cleavage of the primary translation product or the formation of an inactive ASM precursor (Ferlinz, K., et al., (1997) Eur. J. Biochem. Vol. 243, 511-517).
It is generally accepted that a variety of forms of ASM are active in humans, and that these forms are characterized by differing molecular weights and differing glycosylation patterns. ASM has been described in terms of a secretory form found in the circulation, and an intracellular, lysosomal form, both derived from the same gene (Schissel., S. L., et al. (1998) J. Biol. Chem. Vol. 273, 18250-18259). The secretory form, obtained either from fetal bovine serum (Spence, M. W., et al. (1989) J. Biol. Chem. Vol. 264, 5358-5363) or from various cultured cells (Schissel, S. L., et al. (1996), J. Biol. Chem. Vol. 271, 18431-18436), displays increased specific activity in the presence of zinc. Bartelsen et al. also observed copper-dependent activation for recombinant ASM secreted from insect sf21 cells (Bartelsen, O., et al. (1998) J. Biotechnol. Vol. 63, 29-40). The lysosomal form of ASM, however, does not require exogenously added zinc for activation and had been referred to as “cation-independent” (Schissel, S. L., et al. (1996), J. Biol. Chem. Vol. 271, 18431-18436; Levade, T., (1986) J. Clin. Chem. Clin. Biochem. Vol. 24, 205-220). Schissel et al. reported that both the lysosomal and secretory forms can be inactivated by the zinc-specific chelator 1,10-phenanthroline, and thus concluded that both forms require zinc for their enzymatic activity (Schissel., S. L., et al. (1998) J. Biol. Chem. Vol. 273, 18250-18259). This suggested that zinc is already tightly associated with the “cation-independent” lysosomal form, making exogenous zinc unnecessary for maximal activity.
The secretory and lysosomal forms of ASM have been shown to have differences in their glycosylation as well as differences in N-termini (Schissel., S. L., et al. (1998) J. Biol. Chem. Vol. 273, 18250-18259). With regard to post-translational modifications of these two forms, the lysosomal form of ASM has high mannose-type oligosaccharides, required for phosphorylation and lysosomal targeting, while the secretory form contains complex-type N-linked oligosaccharides. The difference in trafficking pathways for the two forms has been proposed as the reason for their different exposure to cellular zinc and thus their difference in zinc sensitivity (Schissel., S. L., et al. (1998) J. Biol. Chem. Vol. 273, 18250-18259). The N-termini of the two forms was shown to be different, due to proteolytic processing of the lysosomal form. Whether differences exist in the C-termini of the two forms has not yet been determined, however C-terminal processing has been reported for several other lysosomal enzymes including acid alpha-glucosidase (Wisselaar., H. A., et al., (1993) J. Biol. Chem. Vol. 268, 16504-16511) and cathepsin D (Yonezawa, S., et al., (1988) J. Biol. Chem. Vol. 263, 2223-2231; Lloyd, J. B., et al. (1996) Subcellular Biochemistry (Harris, J. R., ed) Vol. 27, Plenum Publishing Corp., New York).
It has been proposed that histidine and glutamate residues may participate in metal binding sites within ASM, and comparison of the ASM primary sequence with known zinc metalloproteins suggests as many as seven potential zinc-binding sites (Ferlinz, K., et al., (1997) Eur. J. Biochem. Vol. 243, 511-517). The actual stoichiometry of zinc binding and the specific amino acids responsible for coordination of metal ion within ASM remain to be determined. The status of the 17 cysteine residues within ASM, in terms of disulfide linkages and number of free cysteines, is also not well characterized. It has been shown that dithiothreitol (DTT), but not reduced glutathione, inhibits the enzymatic activity of ASM in a concentration-dependent manner (Lloyd, J. B., et al. (1996) Subcellular Biochemistry (Harris, J. R., ed) Vol. 27, Plenum Publishing Corp., New York). However, the mechanism of this inactivation is not as yet understood. The inactivation may not be simply due to disulfide reduction, as effects of DTT on protein activity unrelated to disulfide reduction have been reported (Lansmann, S., et al. (2003) Eur. J. Biochem. Vol. 270, 1076-1088). In contrast to this inactivation of ASM, lysosomal lipids and the sphingolipid activator protein SAP-C have been shown to stimulate ASM activity (Liu, B., et al. (1997) J. Biol. Chem. Vol. 272, 16281-16287).
As mentioned above, enzyme replacement therapy has been proven to be an effective means of treating some lysosomal storage diseases. With respect to ASM, it has been shown that a recombinant form of the enzyme, expressed in CHO cells, has characteristics consistent with the non-recombinant forms including acid pH optimum, sensitivity to sulfhydryl reducing reagents and inhibition by a zinc specific chelator (Schuchman, E. H., et al. (1992) Genomics Vol 12, 197-205). During the biochemical characterization of purified recombinant human ASM (rhASM) protein, the present inventors discovered that, unexpectedly, the specific activity of the protein increased when the cell harvests were stored frozen at −20° C. for several weeks. This unexpected activation was identified and, as described herein, was determined to involve the C-terminal cysteine residue of ASM, which is present in a number of active forms of human ASM.